US5427939A - Expression vectors encoding alloantigens of glycoprotein Ibα - Google Patents

Expression vectors encoding alloantigens of glycoprotein Ibα Download PDF

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US5427939A
US5427939A US07/817,852 US81785292A US5427939A US 5427939 A US5427939 A US 5427939A US 81785292 A US81785292 A US 81785292A US 5427939 A US5427939 A US 5427939A
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met
platelets
fragment
gpibα
glycoprotein
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Zaverio M. Ruggeri
Jerry L. Ware
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Scripps Research Institute
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Priority to EP93902739A priority patent/EP0625047B1/de
Priority to PCT/US1992/011190 priority patent/WO1993013784A1/en
Priority to ES93902739T priority patent/ES2263147T3/es
Priority to DE69233623T priority patent/DE69233623T2/de
Priority to JP5512464A priority patent/JPH07502744A/ja
Priority to CA002127368A priority patent/CA2127368A1/en
Priority to AU34401/93A priority patent/AU673993C/en
Priority to DK93902739T priority patent/DK0625047T3/da
Priority to AT93902739T priority patent/ATE325192T1/de
Priority to US08/470,137 priority patent/US5798216A/en
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Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: SCRIPPS RESEARCH INSTITUTE
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • This invention relates to providing patients with blood products that minimize recognition and response thereto by the recipient's immune system.
  • the fundamental function of the immune system of the body includes detecting foreign macromolecules that have invaded the body (such as those produced by or attached to a microorganism), distinguishing them from molecules produced by the body (self-molecules), and then producing specific cells and molecules (antibodies) that combine with the foreign macromolecules to inactivate them and eventually destroy them.
  • the immune system functions in part by maintaining a library of cells, each of which is capable of producing a specific antibody that binds to a specific foreign macromolecule, the macromolecule being free in solution or attached to a foreign cell.
  • An antibody is a protein that binds specifically to a foreign macromolecule at an epitope therein, leading to inactivation of the macromolecule that contains the epitope.
  • An epitope is a structural domain or subregion of the macromolecule having a unique structure (for example, charge distribution and shape) that is recognized and targeted by the immune system. Normally, epitopes on self-molecules go unrecognized, that is, the immune system does not identify them as representing foreign structures against which a defense must be made.
  • the immune system In order to function effectively, the immune system must be sensitive, that is, it must be able to detect those differences in structure (which are often very subtle) between "self molecules” and foreign molecules. These differences may include amino acid substitutions in proteins and/or differences in the type or orientation of carbohydrate and lipid components attached to macromolecules, including proteins, glycoproteins, glycolipids and lipoproteins.
  • Platelets are non-nucleated, discoid, cell-like structures which are 2-5 microns in diameter and which are derived from megakaryocytic blood cells. They are crucial to the formation of clots, such as are necessary to heal an injury to a blood vessel, and are often administered to patients to facilitate clot formation.
  • Platelets are believed to participate in clot formation as follows.
  • the restriction or termination of the flow of blood within the circulatory system in response to a wound involves a complex series of reactions which can be divided into two processes, primary and secondary hemostasis.
  • Primary hemostasis refers to the process of platelet plug or soft clot formation. Effective primary hemostasis is accomplished by platelet adhesion, that is, the interaction of a platelet with the surface of damaged vascular endothelium on which are exposed underlying collagen fibers and/or other adhesive macromolecules such as proteoglycans and glycosaminoglycans to which platelets bind; and platelet activation.
  • epitopes against which blood products are typed are: (A) the ABO system that arises from differences in carbohydrate attached to large lipid molecules inserted into the plasma membrane of red blood cells and also of platelets; and (B) the Rh antigens.
  • ABO system that arises from differences in carbohydrate attached to large lipid molecules inserted into the plasma membrane of red blood cells and also of platelets
  • B the Rh antigens.
  • HLA glycoproteins found on the surface of red blood cells and platelets are also recognized by the immune system of transfusion recipients. Immune system response to HLA glycoprotein decreases strongly the likelihood of survival of transplanted organs or of transfused blood products, including platelets. Although proper typing of platelets with respect to HLA glycoprotein has reduced the incidence of platelet transfusion refractoriness, there exist certain additional surface components of platelets wherein variation in the structure thereof between donors and recipient strongly affects the likelihood of a successful program of platelet transfusion.
  • This invention identifies one such structural difference in a platelet surface component and pertains also to the administration of platelet-containing blood products, use of which minimizes adverse immune response in the recipients thereof. There follows hereafter a brief discussion of those factors that determine whether a genetically inherited structural difference in a platelet surface component will trigger an immune response by a transfusion recipient.
  • the differences, such as amino acid substitutions, that define the particular forms of a protein present in different species are much greater than those differences that define the forms of the protein in different individuals of the same species.
  • the immune system is more likely to detect and respond to the differences (reflected in modified epitopes) that exist between species.
  • Genetic variation between individuals of the same species is reflected in "alleles", that is, alternate forms of the gene that encodes a protein.
  • Variation in the amino acid sequence of the protein results from minor differences (mutations) in the coding sequence of the gene.
  • the different forms of the protein encoded by the alternate forms of the gene are termed alloantigens.
  • the immune system may not recognize the difference between the products (alloantigens) of the two alleles.
  • the forms of the protein may be sufficiently different so that antibodies (for example, in a transfusion recipient) can "determine” that one alloantigen is "self” and the other is foreign.
  • Antibodies thus responding to the form of an epitope on one alloantigen, but not to the form of the epitope present on another alloantigen are termed "allotypes". Allotypic antibodies are particularly important in the practice of the invention because it is believed that identification of only a few important alloantigens on platelets is necessary to provide, in general, effective platelet transfusion.
  • This invention defines the locus of a newly discovered platelet alloantigen system, provides for diagnostic screening procedures to detect both forms of the protein, and provides for administration to patients of typed platelets having improved functional characteristics.
  • this invention is associated with the identification of an important locus (amino acid sequence position) on a protein of the platelet surface that is responsible for platelet transfusion refractoriness.
  • the locus is located at residue 145 of platelet glycoprotein Ib ⁇ and is in the form of a threonine/methionine (abbreviated as "Thr/Met”) amino acid dimorphism.
  • a method of inhibiting platelet transfusion refractoriness comprising administering third-party platelets to a patient whose platelet glycoprotein Ib ⁇ polypeptides expressed from the patient's DNA have been determined to have either threonine or methionine residues, or both, at amino acid residue positions 145 thereof, said third-party platelets having expressed at residue position 145 of the glycoprotein Ib ⁇ polypeptide thereof amino acids that are the same as those expressed on the glycoprotein Ib ⁇ polypeptides of the patient's platelets.
  • the invention includes within its scope various strategies whereby potential platelet incompatibility between donor and recipient can be detected and avoided.
  • An important strategy involves the use of an antibody, or a fragment thereof, in substantially pure form having as its epitope a domain of glycoprotein Ib ⁇ , the affinity of said antibody, or fragment thereof, for said epitope being dependent on the identity of the amino acid residue at position 145 of said glycoprotein.
  • the invention provides a supply of blood product comprising platelets segregated into three stocks, one stock containing platelets having at position 145 on glycoprotein Ib ⁇ polypeptide thereof a threonine residue, a second stock containing platelets having at position 145 on glycoprotein Ib ⁇ polypeptides thereof a methionine residue, and a third stock containing platelets having at position 145 on the glycoprotein Ib ⁇ polypeptides thereof, methionine and threonine residues.
  • Another aspect of the present invention encompasses a process for providing a monoclonal antibody directed against a domain of glycoprotein Ib ⁇ , the epitope of said antibody being dependent on the presence of Met 145 or Thr 145 in said glycoprotein, said process comprising: (A) fusing a mixture of lymphocyte cells and myeloma cells to form hybridomas; the source of said lymphocyte cells being lymphocyte cells recovered from an animal that was immunized with glycoprotein Ib ⁇ , or a fragment thereof; (B) isolating a hybridoma that secretes an antibody directed against said epitope; (C) culturing said hybridoma; and (D) collecting the monoclonal antibody produced therefrom.
  • An additional aspect of the invention provides a method of immunoassaying utilizing an antibody, or a fragment thereof, to assay for human glycoprotein Ib ⁇ polypeptides for the presence therein of a Met 145 residue or a Thr 145 residue comprising contacting said antibody with glycoprotein Ib ⁇ polypeptides, or a fragment thereof, and determining whether said glycoprotein Ib ⁇ polypeptides, or fragment thereof, includes a Met 145 residue and/or a Thr 145 residue.
  • Another aspect of the invention provides for the determination of whether there has been administered previously to a patient a platelet-containing blood product containing at position 145 of the glycoprotein Ib ⁇ polypeptide thereof an amino acid residue different from the amino acid residue at position 145 of the patients glycoprotein Ib ⁇ polypeptide. Accordingly, there is provided a method of immunoassaying comprising contacting a sample of blood, or a composition derived therefrom, with alloantigenic Thr or Met 145 form of glycoprotein Ib ⁇ , or a fragment thereof containing said Thr/Met 145 locus, and determining whether there is present a complex of antibody and a form of said glycoprotein or a fragment thereof.
  • the invention includes also within its scope the provision of DNA sequences, expression plasmids, and recombinant host cells through which may be expressed glycoprotein Ib ⁇ polypeptide, or a fragment thereof, containing Met 145 or Thr 145 residues.
  • Thr 145 allele was determined to be present about 89% of the time, whereas the Met 145 form was present at about 11%, leading to a population in which approximately 80% of individuals are homozygous for Thr 145 , 2% are homozygous for Met 145 and 18% are heterozygous. The potential for platelet transfusion refractoriness is thus indicated.
  • FIGS. 1 and 2 show the AhaII restriction pattern of human genomic DNA, encoding in FIG. 1 GPIb ⁇ , containing Thr 145 and/or Met 145 codons.
  • GPIb ⁇ is represented schematically.
  • the GPIb ⁇ precursor protein consists of signal peptide (SP) and a 610-amino acid mature ⁇ -subunit.
  • the mature ⁇ -subunit contains a series of leucine-rich repeats (LRR) within an N-terminal domain in which the binding site for von Willebrand factor is located; it also contains a transmembrane domain (TM) and a cytoplasmic tail.
  • LRR leucine-rich repeats
  • FIG. 2 is a photograph of an agarose gel illustrating the AhaII restriction pattern obtained before (L) and after (R) restriction of human genomic DNA, encoding GPIb ⁇ , containing Thr 145 and/or Met 145 codons.
  • peptide and “polypeptide” are used herein interchangeably. It is understood also that the practice of the invention pertains to the use of human blood products and proteins in human patients.
  • the present invention encompasses also the use of glycoprotein Ib ⁇ polypeptides, or fragments thereof, (and/or the encoding DNA sequences therefor) that may contain, relative to the polypeptides and DNA sequences described herein that reflect the Thr/Met 145 polymorphism, additional mutations which do not affect the biological properties or diagnostic utility of the Thr 145 - or Met 145 -containing alloantigenic epitope of the polypeptides.
  • glycoprotein Ib ⁇ polypeptides of the invention it is also within the scope of the invention to prepare modified forms of the glycoprotein Ib ⁇ polypeptides of the invention, such as by further or different mutation of an encoding DNA therefor, or by derivatization thereof (for example, sulfation, glycosylation, esterification, etc.).
  • GPIb ⁇ polypeptides containing further or additional mutations said GPIb ⁇ polypeptides (such as on donor platelets) are deemed equivalent in the practice of the invention if they have the functional properties of the alloantigen of a recipient and are not recognized by a recipient's immune system as being foreign.
  • the invention encompasses biologically unimportant differences between the actual DNA's and polypeptides utilized in the practice of the invention and the structural sequences of amino acids or nucleotides thereof as reported herein.
  • the threonine/methionine dimorphism at residue 145 of the glycoprotein Ib ⁇ polypeptide of platelets defines an important alloantigen system that is a cause of platelet transfusion refractoriness, that is, an allosensitization to antigens found on platelets, such that additional platelet transfusion does not result in an increased platelet count.
  • glycoprotein Ib ⁇ polypeptide of platelets For background purposes there is set forth hereafter information concerning glycoprotein Ib ⁇ polypeptide of platelets, and its role in clotting (hemostasis).
  • the adhesion of platelets to damaged or diseased vessels occurs through mechanisms that involve specific platelet membrane receptors which interact with specialized adhesive molecules.
  • One such platelet receptor is the glycoprotein Ib-IX complex which consists of a noncovalent association of two integral membrane proteins, glycoprotein Ib (GPIb) and glycoprotein IX (GPIX).
  • GPIb which is a two-chain molecule having an apparent molecular mass of approximately 160 kDa, is composed of a heavy (alpha, or "GPIb ⁇ ") chain, having a molecular mass of approximately 145 kDa, linked by disulfide bonds to a light (beta, or GPIb ⁇ ) chain, having a molecular mass of approximately 22 kDa.
  • GPIb is an integral membrane protein and both the alpha- and beta- chains described above have transmembrane domains.
  • glycocalicin refers to a soluble proteolytic fragment of the heavy ( ⁇ ) chain of GP Ib that is generated by cleavage in a position close to the transmembrane domain of the molecule (Yamamoto, K. et al. Thromb. Res., 43, 41- 55 (1986)). It is now clear that glycoalicin comprises most of the extracellular domains of the GPIb ⁇ from which it derives.
  • the adhesive ligand of the GPIb-IX complex is the protein von Willebrand factor ("vWF") which is found as a component of the subendothelial matrix, as a component of the ⁇ -granules secreted by activated platelets, and also as a circulating blood plasma protein.
  • vWF von Willebrand factor
  • the actual binding site of vWF on the GPIb-IX receptor has been localized on the amino terminal (His 1 -Arg 293 ) region of the ⁇ chain of glycoprotein Ib. This residue 1-293 region of the polypeptide may be generated as a fragment of GPIb ⁇ having a molecular weight of 45 kDa using, for example, trypsin to effect the necessary proteolytic cleavage.
  • vWF-GPIb ⁇ interaction results in the prevention of primary hemostasis and the induction of an anti-thrombotic state useful in prevention of other diseases in which occlusion of blood vessels plays an important role.
  • the interaction of GPIb ⁇ with vWF is believed to be unaffected by the Thr/Met 145 dimorphism of GPIb ⁇ polypeptide that is responsible for the immune phenomena detected in the practice of this invention.
  • the amino acid sequence of the amino terminal 45 kDa fragment of GPIb ⁇ has been reported by Titani, K. et al., Proc. Natl. Acad. Sci. USA, 84, 5610-5614 (1987).
  • GPIb ⁇ A complete cDNA encoding human GPIb ⁇ polypeptide has been determined by Lopez et al., Proc. Natl. Acad. Sci. USA, 84, 5615-5617 (1987).
  • the gene for GPIb ⁇ has been cloned from a genomic cosmid library utilizing a partial cDNA clone as a probe, and its sequence, including introns, has been determined by Wenger, R. H. et al. Biochemical and Biophysical Research Communications, 156(1), 389-395 (1988).
  • the GPIb ⁇ sequence predicted thereby consists of a 16 amino acid signal peptide, Met -16 through Pro -1 , followed by a 610 amino acid mature peptide or polypeptide region, His 1 through Leu 610 .
  • the nucleotide numbering system of Wenger is used herein.
  • the structure and properties of GPIb ⁇ are reviewed in Ruggeri, Z. M., The Platelet Glycoprotein Ib-IX Complex, in Progress in Hemostasis and Thrombosis, vol. 10, p.3568, Coller, B.S. ed., W. B. Saunders Co., Philadelphia, 1991.
  • This invention provides improved techniques of transfusion to a recipient of third-party blood products that contain platelets.
  • the incidence and severity of platelet transfusion refractoriness is minimized following the practice of the invention.
  • Representatives of the many aspects of the invention are three important techniques: (A) a method to determine (to type) the alloantigenic forms of glycoprotein Ib ⁇ of a patient so that the patient receives a transfusion of donor (third-party) platelets containing the identical alloantigenic form of GPIb ⁇ , for the Thr/Met 145 locus thereof; (B) a method of determining whether a patient who would otherwise benefit from third-party platelets (as, for example, in a transfusion) has produced antibody to previously administered platelets with respect to the Thr/Met 145 locus of glycoprotein Ib ⁇ ; and (C) a method of determining in a donor the identity of alloantigenic forms of GPIb ⁇ , for the Thr/Met 145 locus thereof.
  • This flexibility is particularly important for administration of emergency medical treatment when supplies of properly typed and segregated platelets may not be available.
  • the diagnostic procedures of the invention provide warning of restrictions upon the alloantigenic forms (Thr/Met 145 ) which can be safely administered.
  • Typing of third-party (donor) platelets to meet the above goals is similarly accomplished according to the invention.
  • platelets proper typing of platelets is particularly important with respect to treatment of cancer patients, particularly those having a leukemia and subject to chemotherapy and/or bone marrow irradiation and having low levels of platelet production. Such patients require frequent administration of platelets and are at risk of platelet transfusion refactoriness.
  • Example 1 of the invention describes the production of such suitable fragments of glycoprotein Ib ⁇ , including a His 1 -Ala 302 fragment of GPIb ⁇ expressed from mammalian cells, a tryptic fragment of GPIb ⁇ consisting of the 45 kDa residue 1-293 domain of GPIb ⁇ , glycocalicin, and glycosylated and unglycosylated peptides, derived from GPIb ⁇ , that include residue 145 thereof.
  • Thr 145 and Met 145 forms of the His 1 -Ala 302 fragment of GPIb ⁇ expressed as provided in Example 1 mimic the proper three-dimensional conformation of their respective domains as would be present on their respective allelic forms of GPIb ⁇ on platelets.
  • the Met 145 alloantigenic form of GPIb ⁇ provides the antigenic epitope recognized by Sib a antibody.
  • the epitope(s) for Sib a antibody responds to a domain of GPIb ⁇ that includes residue 145 (in methionine form and not in threonine form) or responds instead to another epitope of the molecule (a neoantigenic epitope which is only present in GPIb ⁇ when the relatively rarer Met 145 residue is present causing a conformational change in the molecule that exposes the new domain).
  • Antibody appropriate for the practice of the invention may be generated by any of a number of procedures and can be polyclonal or monoclonal.
  • a preferred reference manual of techniques for producing, screening and characterizing antibodies is Harlow, E., and Lane, D., eds. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988).
  • one preferred approach comprises providing a clone that produces a monoclonal antibody, or providing a set of such clones which produce different monoclonal antibodies that respond to different exact epitopes dependent on Thr 145 or Met 145 .
  • Production of such antibody can be accomplished by procedures well known in the art such as that of Kohler, et al., Eur. J. Immunol., 6, 292-295 (1976) wherein fusion of myeloma cells to animal lymphocytes that have been immunized with an appropriate antigen is accomplished.
  • Suitable myeloma cell lines may be derived from BALB/c mouse MOPC 21 myelomas as described by Kohler, et al.
  • fusion of lymphocytic cells and myeloma cells is effected by adding a suspension of lymphocytic cells to myeloma cells in the growth medium and centrifuging to form a pellet. The cells are then incubated in a growth medium containing the fusing agent. Hybridomas which synthesize and secrete antibodies are then cultured to establish continuously proliferating cell lines in tissue culture. The hybridomas are segregated into individual clones and the antibody is recovered from tissue culture thereof.
  • a preferred reference on the production of monoclonal antibodies is Antibodies: A Laboratory Manual, Harlow, E and Lane, D eds., supra at pages 150-238 thereof.
  • a second strategy for the production of antibodies suitable in the practice of the invention comprises immunizing an animal with a peptide that includes Met or Thr 145 .
  • Use of a small peptide instead of a large glycoprotein minimizes the chance that the immunized animal will make a large fraction of its antibodies to irrelevant epitopes.
  • peptides which contain a Met or a Thr 145 residue it is preferred that such peptides be up to about 30 amino acids in length comprising fragments of GPIb ⁇ in glycosylated or nonglycosylated form.
  • For preferred methodology see Harlow, E., and Lane, D., supra, at pages 72-121 thereof. A polyclonal population of antibody results.
  • a third strategy involves generating a polyclonal response in a rabbit immunized with, for example, the residue 1-302 fragment (as Met 145 ) and using immune depletion of the resultant serum with the Thr 145 form of the fragment.
  • any fragment thereof that contains all or a portion of the variable region thereof and therefore retains the capability of binding to all or part of a GPIb ⁇ epitope include Fab and F(ab') fragments.
  • an antibody, or fragment thereof having a binding affinity constant for GPIb ⁇ , or for a fragment thereof defining the epitope is preferred.
  • the invention provides a method to inhibit platelet transfusion refractoriness in a patient that comprises administering to said patient third-party platelet preparations containing GPIb ⁇ polypeptides to which the patient's immune system does not direct an antibody response.
  • the invention has been described primarily in the context of a dimorphism at residue 145 of GPIb ⁇ , it is noted that additional alleles may come to be known that encode additional amino acid species at residue position 145 and that the general DNA screening procedures of the invention (see, for example, Example 2) may be used to characterize the DNA of such individuals. Similar procedures are effective at all the other amino acid sequence positions of GPIb ⁇ to identify alloantigenic loci and provide clinical procedures to compensate for amino acid polymorphisms that create such "foreign" epitopes.
  • any suitable procedure which allows for the quantitative or qualitative assessment of the binding of an antibody directed to an epitope of GPIb ⁇ dependent upon the Thr/Met 145 locus thereof is effective in the practice of the invention.
  • suitable procedures are outlined in Examples 3 and 4 below.
  • the antigens are available in large amounts, an antibody capture is a convenient way to screen for the required hybridoma cell line. Both antigens are spotted onto nitrocellulose (NC or dot-blot) or applied onto a polyvinylchloride multiwell plate. The NC or plate is blocked with a protein solution of at least 1 ⁇ g/ml. Approximately 1 ⁇ l of hybridoma tissue culture supernatant is added to each square (NC) or 200 ⁇ l is added to the well of each plate. After an incubation for 1 hour, the NC or wells are washed 3 times with the same blocking solution.
  • Bound antibody is detected by a second incubation with an 125 I-labeled rabbit anti-mouse immunoglobulin (using approximately 50,000 cpm per NC spot or well), The wells are then washed 3 times in the same blocking solution and exposed to autoradiography (NC) or to gamma ray counting in a gamma spectrometer. This procedure will allow an identification of clones that are secreting antibodies specific for either the Thr 145 or Met 145 form of GPIb ⁇ .
  • the procedures described above are effective for the purpose of providing antibody useful for determining whether or not a patient has been previously exposed to a third-party blood product containing platelets, the GPIb ⁇ thereof being of different alloantigenic form with respect to the Thr/Met 145 locus thereof than that of the patient.
  • Effective means whereby facilities that provide health care and/or store blood products containing platelets can minimize adverse immune reactions thereto in patients and maximize the effectiveness of platelet-containing transfused products.
  • Such procedures include utilizing the diagnostic reagents provided by the invention to type donors and recipients, and to segregate supplies of platelets based on identification of the amino acid residue at position 145 of the GPIB ⁇ thereof.
  • a blood bank or medical care facility should segregate blood products containing platelets into different stocks, one stock containing only platelets with GPIb ⁇ having the Thr 145 form thereof, a second stock containing platelets having only the Met 145 form thereof, and (optionally) a third stock may be prepared containing both the methionine and threonine 145 forms of GPIb ⁇ polypeptides and being suitable for administration to heterozygous individuals.
  • Therapeutic use of platelet stock so segregated comprises a preferred method of inhibiting hemorrhage in a patient in that the above blood products will remain effective in the patient for longer periods than products as presently administered, surviving, for example, for a longer time without being removed from circulation by the kidneys or becoming, as a result of immunological reactions, otherwise inoperable in a patient.
  • GPIb ⁇ polypeptide and/or fragments thereof suitable as alloantigen for the production of allotypic antibody
  • Elements necessary for the preparation of the suitable antigenic polypeptides of the invention are: (A) DNA sequences which encode the residue His 1 -Leu 610 , His 1 -Ala 302 , or similar domains of the GPIb ⁇ polypeptide that contain the epitope(s) that reflect the Thr/Met 145 dimorphism; (B) an expression plasmid or viral expression vector capable of directing in a eucaryotic cell the expression therein of the aforementioned domains; and (C) a eucaryotic host cell in which said expression may be effected.
  • the GPIb ⁇ polypeptides so expressed are expected not to be secreted from host cells because of the lack of attachment to the nascent GPIb ⁇ polypeptide of a signal peptide. Purification of proteins expressed therein and the extraction of pharmacologically useful quantities thereof are expected to be more difficult than if the polypeptide could be caused to be secreted into the culture medium of the host cells.
  • Signal peptides corresponding to other protein species may prove equally effective to cause the secretion of GPIb ⁇ . von Heijne, G., J. Mol. Biol., 184, 99-105 (1985).
  • the signal peptide When attached to the amino terminal end of the residue 1-610 or 1-302 GPIb( ⁇ ) polypeptide, the signal peptide causes the polypeptide to be recognized by cellular structures as a polypeptide of the kind to be processed for ultimate secretion from the cell, with concomitant cleavage of the signal polypeptide from the mature GPIb ⁇ polypeptide.
  • a wide variety of expression plasmids or viral expression vectors are suitable for the expression of the GPIb ⁇ polypeptides or the amino terminal regions thereof.
  • One factor of importance in the selection of an expression system is the provision of a high efficiency transcription promoter directly adjacent to the cloned GPIb ⁇ insert.
  • Another factor of importance in the selection of an expression plasmid or viral expression vector is the provision of an antibiotic resistance gene marker therein so that continuous selection for stable transformant eucaryotic host cells can be applied.
  • Plasmids suitable in the practice of the invention include pCDM8, pCDM8 neo , pcDNA1, pcDNA1 neo , pMAM neo and Rc/CMV. Plasmids whose use in the practice of the invention is preferred include pCDM8 neo , pcDNA1 neo , pMAM neo and Rc/CMV. A DNA sequence encoding the GPIb ⁇ polypeptide, or a fragment thereof, may also be inserted into a plasmid or vector suitable for causing expression of the polypeptide in a bacterial system.
  • viral expression vector systems in the practice of the invention, including for example, those based upon retroviruses and those based upon baculovirus Autoqrapha californica nuclear polyhidrosis virus.
  • Representative host cells comprising permanent cell lines suitable for the practice of the invention include CHO-K1 Chinese hamster ovary cells, ATCC-CCL.61; COS-1 cells, SV-40 transformed African Green monkey kidney, ATCC-CRL 1650; ATT 20 murine pituitary cells; RIN-5F rat pancreatic ⁇ cells; cultured insect cells, Spodoptera frugiperda; or yeast (Sarcomyces).
  • Thr 145 form of the 45 kDa fragment or of glycocalicin may be derived from individuals homozygous for GPIb ⁇ containing Thr 145 (corresponding to the first discovered allele, Lopez, J. A. et al. Proc. Natl. Acad. Sci. USA,. 84, 5615-5617 (1987)).
  • Met 145 forms of the above fragments may be prepared from GPIb ⁇ of individuals homozygous for the Met 145 form.
  • Polypeptides of heterozygous individuals may be separated using, for example, immunoaffinity chromatography sensitive to the relevant difference in epitope or conformation.
  • Synthetic peptides containing up to about 30 amino acids, and comprising fragments of glycoprotein Ib ⁇ (containing a residue position equivalent to either Met 145 or Thr 145 ) can be prepared, for example, by following the method of Houghten, R. A., Proc. Natl. Acad. Sci. USA, 82, 5131-5135 (1985) with further purification as described in Vicente, V. et al., J. Biol. Chem., 265(1), 274-280 (1990). Glycosylation may be added to such synthetic peptides by coupling at appropriate sites to mimic natural glycosylation.
  • sufficient quantities of peptide to serve as antigen may be derived from other fragments of GPIb ⁇ of blood products, produced therefrom, for example, by multiple proteolytic cleavages and subsequent purification, for example, by high pressure liquid chromatography.
  • Genomic DNA was isolated, after informed consent, from the peripheral blood lymphocytes of healthy blood donors recruited at the General Clinic Research Center facility of the Scripps Clinic, La Jolla, Calif., following procedures well known in the art. See Blin, N. et al., Nucleic Acid Res., 3, 2303 (1976). The lack of introns within the coding sequence for GPIb ⁇ was exploited to permit amplification of genomic DNA fragments of the GPIb ⁇ gene to determine the allelic character of the residue 145 locus and to identify the genetic basis of the Sib a antigen. Two oligonucleotide primers, Ib ⁇ -3 (5'-GGACG . . .
  • CGGC-3' corresponding to GPIb ⁇ residues 106-112, and representing nucleotides 898-918 according to the system of Wenger, R. H. et al. and also Ib ⁇ -4 (5'-GCTTT . . . TGAC-3') corresponding to GPIb ⁇ residues 296-302, and representing nucleotides 1470-1489 were used in a polymerase chain reaction (PCR) to generate a 591 base pair fragment that corresponds to the GPIb ⁇ -encoding sequence for polypeptide residues 106-302.
  • PCR polymerase chain reaction
  • the DNA fragment was amplified using a DNA thermal cycler (Perkin Elmer-Cetus, Berkeley, Calif.) in a final volume of 100 ⁇ l containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.3 mM MgCl 2 , 0.01% gelatin, 0.2 mM of each deoxynucleotide triphosphate, 0.5 ⁇ g of genomic DNA and 2.5 U Taq polymerase.
  • the fragment was generated from a PCR comprising 30-cycles consisting of 94° C. for 30 seconds (s), 52° C. for 30 s, and 72° C. for 60 s. Cloning of amplified fragments into M13mp18 bacteriophage, and subsequent DNA sequence analysis were performed using standard techniques.
  • FIG. 1 schematically illustrates the PCR strategy used to generate a 591 base pair (bp) fragment of the GPIb ⁇ gene that codes for amino acid residues 106-302, and restriction thereof.
  • the Thr/Met 145 amino acid dimorphism is the result of a single nucleotide transition changing the Thr codon (ACG) to a Met codon (ATG).
  • genotype of any individual at this locus can be determined by restriction enzyme digestion of amplified genomic DNA since the identified nucleotide transition (Thr ⁇ Met) destroys the AhaII restriction site, 5'-G(G/A)CG(T/C)C-3', (nucleotides 1016-1021, GACGCC to GATGCC) within the GPIb ⁇ -encoding DNA.
  • the polymorphism may be analyzed by the restriction pattern of AhaII (FIG. 2).
  • genomic DNA from 61 healthy blood donors was subjected to PCR analysis and digestion with AhaII.
  • a representative agarose gel analysis (FIG. 2) shows the observed genotypes.
  • the expected three genotypes related to codon 145 were observed.
  • Some individuals contained two alleles coding for Met 145 , some contained two alleles coding for Thr 145 , and some were heterozygous containing one allele coding for Met 145 and one allele coding for Thr 145 .
  • the cumulative results revealed allele frequencies of 89% and 11% for the Thr 145 and Met 145 codons respectively.
  • the allele frequencies within a population of restricted ethnic origin may differ from those reported herein.
  • PCRs were also performed on genomic DNA isolated from several healthy individuals whose platelets reacted with the anti-Sib a antibody.
  • the amplified DNA fragments were cloned into M13mp18 for DNA sequence analysis.
  • the sequence analysis revealed a cytosine to thymine transition at nucleotide position 1018 in several of the cloned fragments from these individuals.
  • the observed nucleotide transition demonstrates that the threonine 145 ACG codon, matching the reported GPIb ⁇ sequence, is substituted by a methionine 145 ATG codon in individuals showing this antibody response.
  • Serum containing the anti-Sib a antibody used in this study was obtained from the original propositus reported by Saji, H. et al., Vox Sang, 56, 283 (1989), and was a gift of Dr. Hiroh Saji, Kyoto Red Cross Blood Center, Kyoto, Japan. Platelet reactivity with the anti-Sib a antibody was evaluated using a modified antigen capture ELISA procedure. Ichida, F. et al., Blood, 78, 1722 (1991).
  • washed platelets (1 ⁇ 10 8 /ml) to be tested were mixed with 5-20 ⁇ l of the anti-Sib a serum for 1 hour at 22°-25° C., and the platelets were lysed with "PBS", phosphate buffered saline consisting of 10 mM, Na 2 HPO 4 , 0.14M NaCl, and containing also 10 mM EDTA and 1% Triton X-100.
  • the lysate was centrifuged (11,750 g) to remove the insoluble debris and the supernatant was incubated (60 min) in a microtiter plate coated with the anti-GPIb-IX monoclonal antibody SZ1.
  • the SZ1 antibody (purchased from Immunotech, Marseille, France) is directed against an epitope in the human GPIb-IX complex and was coated previously into the microtiter plate wells by an overnight incubation (3 ⁇ g/ml) at 4° C., followed by washing with PBS containing also 0.05% Tween 20.
  • the amount of anti-Sib a -antibody/Sib a -antigen immune complex bound to SZ1 was determined using a biotinylated goat anti-human IgG (Tago, Burlingame, Calif.) and subsequent incubation with alkaline phosphatase-streptavidin (Zymed, San Francisco, Calif.).
  • Sib a antigen has been determined (see Example 2 above) to represent the GPIb ⁇ polypeptide including Met 145 at the Thr/Met 145 locus thereof
  • other antibodies besides Sib a that are directed to an epitope of GPIb ⁇ dependent on the presence of a Met 145 or Thr 145 residue in said polypeptide may be used similarly in the assay.
  • Such antibodies may be a monoclonal antibody or a a polyclonal population such as from human serum.
  • the total amount of GPIb ⁇ antigen was measured by determining its reactivity with a specific rabbit antiserum obtained by immunization with a synthetic peptide corresponding to GPIb ⁇ residues Gly 271 -Glu 285 (Vicente, V. et al., 1990). Use of this epitope allows quantization of antigen without reference to binding affinity at, or because of, Thr 145 or Met 145 .
  • Blotto a solution composed of 50 mg/ml fat-free milk, 0.25 mM phenylmethylsulfonyl fluoride, 0.15M NaCl, in PBS.
  • Blotto a solution composed of 50 mg/ml fat-free milk, 0.25 mM phenylmethylsulfonyl fluoride, 0.15M NaCl, in PBS.
  • the membrane was washed 3 times in Blotto, incubated for 1 hour with 125 I-labelled goat anti-rabbit IgG, and washed again 3 times in Blotto.
  • Discs of the nitrocellulose membrane corresponding to the position of each application well were cut out and the amount of bound radioactivity was determined with a ⁇ -scintillation spectrometer.
  • the amount of radioactivity bound to each application well was proportional to the amount of GPIb ⁇ antigen bound to the nitrocellulose membrane. Based on the results of this assay, culture media from CHO-K1 cells in which were present residue 1 to 302 fragments of GPIb ⁇ (as Thr or Met 145 forms) were diluted (normalized) to obtain identical amounts of GPIb ⁇ antigen.
  • culture media were tested for their reactivity with the anti-Sib a antibody (see Example 3) using the same dot blot technique.
  • Culture media were vacuum-drawn through a nitrocellulose membrane, blocked with Blotto for 2 hours at 22°-25° C. and then incubated for 2 hours with serum containing the anti-Sib a antibody or normal serum (1:20 dilutions).
  • Tris-buffered saline (10 mM Tris, pH 7.5, 140 mM NaCl) containing also 5% fat-free milk
  • the membrane was transferred into a solution of horseradish peroxidase-conjugated goat anti-human IgG (1:500, Zymed, San Francisco, Calif.) for 2 hours.
  • the membrane was finally washed 3 times with Tris-buffered saline and incubated in a solution containing O-phenylendiamine (Zymed) and hydrogen peroxide to develop color, following manufacturer's instructions.
  • Sib a antigen has been determined (see Example 2 above) to represent the GPIb ⁇ polypeptide including Met 145 at the Thr/Met 145 locus thereof
  • other antibodies besides Sib a that are directed to an epitope of GPIb ⁇ dependent on the presence of a Met 145 or Thr 145 residue in said polypeptide may be used similarly in the assay.
  • Such antibodies may be a monoclonal antibody or a polyclonal population, such as from human serum.
  • the antigenic properties of other fragments of GPIb ⁇ , expressing epitope dependent on the Thr/Met 145 locus can be similarly assayed as long as they adhere to the nitrocellulose and be recognized thereon by antibody.
  • a recombinant expression plasmid, pMW2 was constructed to contain a partial GP Ib ⁇ sequence (His 1 -Ala 302 ) essentially corresponding to the extracytoplasmic 45 kDa domain of the molecule (His 1 -Arg 293 ; Titani et al., 1987).
  • pMW2 a DNA fragment corresponding to nucleotides 503-1490 of the GP Ib ⁇ gene (Wenger et al., 1988) was synthesized in a polymerase chain reaction that added BamHI restriction sites on the ends of the amplified fragment.
  • the synthetic BamHI fragment was cloned into M13 mp19 and sequence analysis verified a coding sequence identical to the corresponding region of the GP Ib ⁇ cDNA (Lopez et al., 1987).
  • the GP Ib ⁇ insert of the M13 construct was recloned as an EcoRI-XbaI fragment into the bacterial plasmid pBS/KS- (Stratagene). This strategy was possible as a result of the initial cloning into M13 mp19 and the acquisition of EcoRI and XbaI restriction sites from the M13 polylinker sequence.
  • the GP Ib ⁇ insert of the pBS/KS- construct was finally cloned into the plasmid pCDM8 neo , and the resultant recombinant molecule was designated pMW2.
  • Cloning of the GP Ib ⁇ fragment from the pBS/KS- construct into pCDM8 neo utilized two unique restriction sites acquired from the pBS/KS-polylinker sequence; an XhoI site located 5' to the GP Ib ⁇ initiating methionine codon and a NotI site located 3' to the codon for mature GP Ib ⁇ residue 302.
  • pCDM8 neo contains a neomycin resistance gene within the eukaryotic expression plasmid pCDM8 (Seed 1987).
  • the expression construct, pMW2 contains coding sequence that predictably synthesizes a primary translation product containing the GP Ib ⁇ signal peptide, the sequence of the mature protein between residues 1 and 302, and an additional 7-residue COOH-terminal sequence, unrelated to GP Ib ⁇ and preceding the first in-frame termination codon, TAG.
  • CHO-K1 Chinese hamster ovary cells
  • CHO-K1 Chinese hamster ovary cells
  • GIBCO heat-inactivated fetal calf serum
  • Cells from confluent cultures were harvested with 0.25% trypsin, 0.2% EDTA, grown for 1 day in 60-mm dishes (1.2 ⁇ 10 5 cells/dish seeding density) and transfected with 10 ⁇ g of the appropriate plasmid DNA using the calcium phosphate-mediated transfection procedure (Chen and Okayama, 1987).
  • cells were harvested with trypsin-EDTA and 1.2 ⁇ 10 4 cells were plated on a 60-mm dish. Cells were cultured for 14 days in medium containing 10% fetal calf serum, L-glutamine, nonessential amino acids, and 800 ⁇ g/ml of Geneticin (Sigma). Independent clones were picked using cloning rings and grown in 12-well plates in medium containing Geneticin for 7 days.
  • LJ-P3 Two different anti-GPIb ⁇ antibodies were used in dot-blot analysis for the identification of clones expressing recombinant protein: LJ-P3, that detects a native conformation-dependent epitope in the amino-terminal 45 kDa tryptic fragment of the protein (Handa et al., 1986), and LJ-Ib ⁇ 1, which reacts with an epitope located between residues 1-237 and is not dependent on the native conformation of the protein.
  • the antibody reacts more strongly with SDS-denatured and reduced GP Ib ⁇ than native GP Ib ⁇ (Vicente et al., 1988).

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